Changliang Zhu

Cytochrome P450 genes expressed in the deltamethrin-susceptible and -resistant strains of Culex pipiens pallens

cDNA sequences encoding cytochrome P450 were amplified, respectively, from deltamethrin-susceptible and -resistant strains of the mosquito, Culex pipiens pallens, with a pair of degenerate primers by RT-PCR and the Direct Cloning Method, and then identified by cDNA chip and Northern blot. Twenty-four positive clones obtained were shown to be new sequences. They had been placed in GenBank and were approved by the Cytochrome P450 Nomenclature Committee, classified to the subfamilies CYP4C, CYP4D, CYP4H, and CYP4J. The microarray signal values of five P450 sequences (CYP4H21, 4H22v1, 4J4v2, 4J6v1, and 4J6v2) were 3.1–7.7 times higher in resistant probe than in susceptible probe and the sequence 4H23v2 reacts only with resistant probe. The results of Northern blot of the above six P450 sequences were similar to that of microarray analyses. These results indicate that CYP4 may be related to deltamethrin resistance in Cx. pipiens pallens.

Larvicidal activity of extracts of Ginkgo biloba exocarp for three different strains of Culex pipiens pallens

Abstract Ethanolic extracts from the Ginkgo biloba L. exocarp from the Chinese ginkgo were assayed against larvae of three strains of Culex pipiens pallens Coquillett. The chemical compositions were detected using a Hewlett–Packard 6890/5973 mass spectrometric detector. The larvicidal bioassay was carried out according to the recommendations of the World Health Organization. The analysis of the essential oil of ginkgo exocarp showed that its major components are ginkgo acid (85.3%) and ginkgo phenolic (5.69%). The larvicidal bioassay showed that extracts of ginkgo exocarp have LC50 of 18.6, 12.7, and 25.0 mg/liter for deltamethrin-susceptible, deltamethrin-resistant, and field strains, respectively. The acute toxicity concentrations of the ginkgo extracts that killed 50% (LD50) of Wistar rats within 2 wk and young carp within 96 h were 4947.2 mg/kg and 557.9 mg/liter, respectively. These results are promising in creating new, effective, and affordable approaches to mosquito control.

Cloning and characterization of myosin regulatory light chain (MRLC) gene from Culex pipiens pallens

Myosin regulatory light chain (MRLC) (GenBank accession no. DQ140391) was cloned from Culex pipiens pallens. An open reading frame (ORF) of 630 bps was found to encode a putative 210 amino acids protein which shows 73% similarity with myosin regulatory light chain of Gryllotalpa orientalis. Real-time quantitative PCR analysis demonstrated that the transcription level of MRLC in deltamethrin-resistant strain (DR-strain) was 4.08-fold higher than in deltamethrin-susceptible strain (DS-strain) of C. pipiens pallens. Over-expression of MRLC in Aedes albopictus C6/36 cells conferred protection against deltamethrin based on tritiated methyl tritiated thymidine ((3)H-TdR) incorporation assay. These results indicate that MRLC may be a potential cause of deltamethrin resistance in C. pipiens pallens.

Cloning and characterization of 60S ribosomal protein L22 (RPL22) from Culex pipiens pallens.

The 60S ribosomal protein L22 (GenBank accession no. EF990190) was cloned from Culex pipiens pallens. An open reading frame (ORF) of 447 bps was found to encode a putative 148 amino acids protein which shares 90% and 80% identity with RPL22 genes from Aedes aegypti and Anopheles gambiae respectively. Real-time quantitative PCR analysis demonstrated that the transcription level of RPL22 in deltamethrin-resistant strain was 2.57 folds higher than in deltamethrin-susceptible strain of Cx. pipiens pallens. Overexpression of RPL22 in C6/36 cells showed that the deltamethrin-resistance was decreased in C6/36-RPL22 cell compared to the control. The mRNA level of cytochrome P450 6A1 (CYP6A1, GenBank accession no. FJ423553) showed that CYP6A1 was down-regulated in the C6/36 transfected with RPL22 (C6/36-RPL22) cells, suggesting that CYP6A1 was repressed by RPL22. Our study provides the first evidence that RPL22 may play some role in the regulation of deltamethrin-resistance in Cx. pipiens pallens.

Partial characterization of deltamethrin metabolism catalyzed by chymotrypsin

Deltamethrin degradation was assessed by measuring deltamethrin reduction with GC-MS and UV-Vis spectrophotometry following incubation of various concentrations (4.96-24.80 microM) of deltamethrin with alpha-chymotrypsin from bovine pancreas. The retention times of crude products of deltamethrin metabolic reactions were 37.968 min, 37.415 min, 36.490 min, and 35.895 min. The UV-Vis spectrophotometric peak absorbance of deltamethrin was at 264 nm, and the peak absorbance of deltamethrin metabolic products were at 250 nm and 296 nm, respectively. Michaelis-Menten metabolic rate constants (V(max) and K(m)) were calculated by nonlinear regression for chymotrypsin using GraphPad Prism 4.0, the V(max) was 97.97+/-26.57 nmol/L/min and K(m) was 7.84+/-3.83 microM. The larvicidal bioassay tests indicated that the mixture from the degradation reaction showed LC(50) increased significantly (P<0.05). This is the first report demonstrating deltamethrin metabolism by chymotrypsin.

Continuous, mixed, and alternating exposure to deltamethrin and fenthion affect development of resistance in Culex Pipiens Pallens in China

ABSTRACT A field population of Culex pipiens pallens was collected from Nanjing, China on July in 2000 and reared in an insectarium. Larvae were subjected to single, mixed, and alternating exposure to deltamethrin and/or fenthion, and the surviving early 4th instars were reared for establishment of adult colonies. Larvae from the colonies were then subjected to the same selection pressures over the subsequent 15 generations. Resistance rates and ratios were measured as LC50 values derived from larval bioassays. In populations exposed to deltamethrin or fenthion alone (single exposure), resistance levels rose rapidly. The LC50 values for deltamethrin and fenthion alone were 29.3 and 1.565 mg/liter, respectively, and the ratios of resistance were 697.6- and 24.8-fold, respectively. Exposure to a mixture of deltamethrin and fenthion (1:1; mixed selection) reduced the development of resistance. The LC50 value and ratio of resistance for the mixture of deltamethrin and fenthion were 0.607 mg/liter and 14.8-fold, respectively, at generation 15. Exposure to alternating treatments of deltamethrin and fenthion (alternating selection) showed an even lower development of resistance. For the alternating treatments, the LC50 value and ratio of resistance to deltamethrin were 0.795 mg/liter and 17.7-fold, respectively (generation 14), and those to fenthion were 0.219 mg/liter and 3.6-fold, respectively (generation 15). Together, these results indicate that the single continuous insecticide selection generated a much more severe resistance than a mixture and/or alternating treatments.

Identification of protease m1 zinc metalloprotease conferring resistance to deltamethrin by characterization of an AFLP marker in Culex pipiens pallens

BackgroundContinuous and excessive application of deltamethrin (DM) has resulted in the rapid development of insecticide resistance in Culex pipiens pallens. The quantitative trait loci (QTL) responsible for resistance to DM had previously been detected in Cx. pipiens pallens. But locating the QTLs on the chromosomes remained difficult. An available approach is to first characterize DNA molecular markers linked with the phenotype, and then identify candidate genes.MethodsIn this study, the amplified fragment length polymorphism (AFLP) marker L3A8.177 associated with the QTL, was characterized. We searched for potential candidate genes in the flank region of L3A8.177 in the genome sequence of the closely related Cx. pipiens quinquefasciatus and conducted mRNA expression analysis of the candidate gene via quantitative real-time PCR. Then the relationship between DM resistance and the candidate gene was identified using RNAi and American CDC Bottle Bioassay in vivo. We also cloned the ORF sequences of the candidate gene from both susceptible and resistant mosquitoes.ResultsThe genes CYP6CP1 and protease m1 zinc metalloprotease were in the flank region of L3A8.177 and had significantly different expression levels between susceptible and resistant strains. Protease m1 zinc metalloprotease was significantly up-regulated in the susceptible strains compared with the resistant and remained over-expressed in the susceptible field-collected strains. For deduced amino acid sequences of protease m1 zinc metalloprotease, there was no difference between susceptible and resistant mosquitoes. Knockdown of protease m1 zinc metalloprotease not only decreased the sensitivity of mosquitoes to DM in the susceptible strain but also increased the expression of CYP6CP1, suggesting the role of protease m1 zinc metalloprotease in resistance may be involved in the regulation of the P450 gene expression.ConclusionOur study represents an example of candidate genes derived from the AFLP marker associated with the QTL and provides the first evidence that protease m1 zinc metalloprotease may play a role in the regulation of DM resistance in Cx. pipiens pallens.

A study on producing monoclonal antibody with one diagnostic marker screened electrophoretically from the urine of individuals infected with Schistosoma japonicum.

One diagnostic marker (a glycoprotein with 30kDa) from the urine of individuals infected with S. japonicum was screened with electrophoresis, and used as antigen to produce McAbs. Two lines of McAbs, named NP56 and NP54,were obtained. McAb NP56 was of better immunoreactivity and its immunoglobulin isotype was IgG(2b.) McAb NP56 could react with SEA (soluble egg antigen) and AWA (adult worm antigen) and express on miracidia in eggs of S. japonicum. When determined with NP56 as a probe in indirect ELISA, the sensitivity and specificity of positive urine samples (EPG median 69) was 50% and 80% in original urine samples, and 90% and 100% in concentrated urine samples, respectively. The results above indicated that with established means it is feasible to produce McAb with the diagnostic marker screened electrophoretically from the metabolic product of the individuals infected with this pathogen, and that McAb-NP56 is of potential value in the diagnosis of chronic cases and individuals with light infection with S. japonicum.

Molecular cloning and preliminary function study of iron responsive element binding protein 1 gene from cypermethrin-resistant Culex pipiens pallens

BackgroundInsecticide resistance jeopardizes the control of mosquito populations and mosquito-borne disease control, which creates a major public health concern. Two-dimensional electrophoresis identified one protein segment with high sequence homology to part of Aedes aegypti iron-responsive element binding protein (IRE-BP).MethodRT-PCR and RACE (rapid amplification of cDNA end) were used to clone a cDNA encoding full length IRE-BP 1. Real-time quantitative RT-PCR was used to evaluate the transcriptional level changes in the Cr-IRE strain Aedes aegypti compared to the susceptible strain of Cx. pipiens pallens. The expression profile of the gene was established in the mosquito life cycle. Methyl tritiated thymidine (3H-TdR) was used to observe the cypermethrin resistance changes in C6/36 cells containing the stably transfected IRE-BP 1 gene of Cx. pipiens pallens.ResultsThe complete sequence of iron responsive element binding protein 1 (IRE-BP 1) has been cloned from the cypermethrin-resistant strain of Culex pipiens pallens (Cr-IRE strain). Quantitative RT-PCR analysis indicated that the IRE-BP 1 transcription level was 6.7 times higher in the Cr-IRE strain than in the susceptible strain of 4th instar larvae. The IRE-BP 1 expression was also found to be consistently higher throughout the life cycle of the Cr-IRE strain. A protein of predicted size 109.4 kDa has been detected by Western blotting in IRE-BP 1-transfected mosquito C6/36 cells. These IRE-BP 1-transfected cells also showed enhanced cypermethrin resistance compared to null-transfected or plasmid vector-transfected cells as determined by 3H-TdR incorporation.ConclusionIRE-BP 1 is expressed at higher levels in the Cr-IRE strain, and may confer some insecticide resistance in Cx. pipiens pallens.

Cloning and sequence analysis of β-actin gene from Aedes albopictus (Diptera: Culicidae)

Objective: To obtain the complete β-actin gene from Aedes albopictus. Methods: Total RNA was extracted from C6136 cells. Degenerate primers were designed based on the β-actin sequences of An. gambiae. Ae. aegypti, Cx. pipiens pallens and D. melanogaster. By RT-PCR, the product was amplified, purified, cloned into the pGT vector and sequenced. The β-actin sequence was aligned and phylogenetically analyzed by the BLAST program and the CLUSTAL W program. Results: A sequence of 1132 bp including an open reading frame of 1131 bp was obtained (GenBank DQ657919). The deduced protein had 376 amino acids. Aligned to SWISS-PROT, it exhibited a high level of identity with-actins from Anopheles, Drosophila and Culex at the amino acid sequence level. Phylogenetic analysis indicated that Ac. albopictus-actin was much more homologous with invertebrate-actin than with vertebrate β-actin. Conclusion: The gene may he used as the internal control in the experiments of Ac. albopictus.